Chemokines are primarily known for their roles in inflammatory and immune responses. However, these small cytokines have also been shown to be upregulated during development of the granulation tissue of wounds and to act on endothelial cells and keratinocytes, cells that perform important functions in the development of the healing tissue (Martins-Green and Bissell, 1990; Nanney et al., 1995; Martins-Green and Feugate, 1998; Luster et al., 1998). In addition to these cell types, fibroblasts are also critical players during formation of the granulation tissue. These cells are activated by cytokines and growth factors that are released from platelets and produced by macrophages. The activated fibroblasts proliferate and migrate across the provisional matrix formed by the fibrin-plasma fibronectin clot. As the clot is digested by plasmin, fibroblasts replace it with cellular fibronectin, tenascin and collagen III (Mackie et al., 1988; Clark, 1993), molecules that are critical for migration of endothelial cells and keratinocytes into the wound and for proper scar formation. Some of the fibroblasts infiltrating the wound differentiate to become myofibroblasts (Gabbiani, 1996; Powell et al., 1999), cells that have bundles of α-smooth muscle actin (α-SMA) and can contract, contributing to closure of the wound (Germain et al., 1994; Lanning et al., 2000).
Chemokines are small, positively charged, secreted proteins that consist of an N-terminal region of variable conformation, followed by a loop, three antiparallel beta strands and a C-terminal alpha helix (Clark-Lewis et al., 1995). They can be divided into four families based on the position of the first two cysteines. The two major families are the CXC family in which the two cysteines are separated by any single amino acid [e.g. cCAF, IL-8, gro-α/MGSA, SDF-1, PF4, IP-10] and the CC family in which the two cysteines are adjacent [MCPs, RANTES, Eotaxin, MIPs] (Prieschl et al., 1995; Bazan et al., 1997; Zlotnik et al., 1999). These proteins have no modifications other than two disulfide bonds and are multifunctional. Chemokines function in a very tightly regulated dose- and time-dependent manner, strongly suggesting that their actions are affected by the microenvironmental conditions (Dunleavy and Couchman, 1995; Gharaee-Kermani et al., 1996; Rennekampf et al., 1997; Youngs et al., 1997).
The first evidence that chemokines are associated with healing was reported in 1990 when it was shown that the chemokine cCAF (chicken Chemotactic and Angiogenic Factor) is overexpressed during wound healing (Martins-Green and Bissell, 1990). This chemokine is highly expressed in the first 24-48 hours after injury and remains elevated for at least 16 days after wounding (Martins-Green and Bissell, 1990; Martins-Green at el., 1992; Martins-Green and Hanafusa, 1997). It is primarily expressed by the fibroblasts of the granulation tissue, especially where interstitial collagen is abundant and by the endothelial cells of microvessels of the granulation tissue (Martins-Green and Bissell 1990; Martins-Green at al., 1992).
In the chicken chorioallantoic membrane (CAM) assay, at low doses, cCAF is chemotactic for monocyte/macrophages and lymphocytes and after several days of exposure to this chemokine, the ectoderm of the CAM becomes thickened and a granulation-like tissue develops beneath the cCAF-containing pellet. In this granulation-like tissue, there is an increase in the amount of interstitial collagen, and the fibroblasts in the mesoderm are consistently aligned with the collagen fibers and appear to cause tissue contraction (Martins-Green and Feugate, 1998). At high concentrations, however, cCAF stimulates blood vessel sprouting from the preexisting vessels of the CAM (angiogenesis) in the absence of leukocyte chemotaxis (Martins-Green and Feugate, 1998).
Other CXC chemokines have also been associated with wound healing events. For example, in bum wounds, gro-α/MGSA is expressed by keratinocytes as they differentiate after re-epithelialization of the wound. Furthermore, CXCR2, the receptor for MGSA, is present in migrating and proliferating keratinocytes (Nanney et al., 1995; Rennekampff et al., 1997). In the granulation tissue, MGSA expression is associated with fibroblasts, smooth muscle cells/myofibroblasts and a subpopulation of macrophages (Nanney et al., 1995). This pattern of expression strongly suggests a role of this chemokine in healing of burn wounds. It also has been shown that in transgenic mice expressing IP-10, a CXC chemokine that inhibits angiogenesis, wounds heal poorly and exhibit defects in development of the granulation tissue (Luster et al., 1998).
It is becoming increasingly more evident that chemokines are expressed at the sites of injury and that they affect processes involving proper development of the granulation tissue. Fibroblasts are critical participants in the development of this healing tissue and they also express high levels of chemokines upon stimulation by stress-inducing agents such as those released upon wounding. Despite this correlative evidence, little is known about how these small cytokines affect wound fibroblast function.